The main objective of the proposed research is to elucidate some of the mechanisms for the control and regulation of specific DNA-directed RNA transcription in eucaryotic cells. Regulation of RNA transcription from the chloroplast genome of Euglena gracilis will be studied at defined physiologic stages such as during chloroplast development, synchronous cell growth, and chloroplast development blocked by mutation or inhibitors. In previous work a transcription complex composed of chloroplast DNA, the associated chloroplast RNA polymerase which is actively synthesizing RNA from the chloroplast DNA template, and other proteins has been purified in high yield from Euglena chloroplasts. This preparation gives evidence of synthesizing chloroplast specific RNA in vitro. Isolation of this complex allows the study of RNA synthesis in a highly purified system. The RNA polymerase of the complex will be purified to characterize its properties and to compare the nature of the RNA product from the homogenous enzyme and chloroplast DNA to that of the intact transcription complex. Then reconstitution of other purified proteins from the complex will be used to characterize the minimum regulatory system required to duplicate the in vivo transcript from the chloroplast genome. The in vitro and in vivo RNA transcripts will be characterized and compared by size analysis, base composition, and DNA-RNA hybridization. Analysis of the kinetics of hybridization of excess chloroplasts RNA to separated single strand and duplex 125I- chloroplast DNA will be employed to determine the percent of the chloroplast genome expressed, the degree of asymmetry of the transcript, and the relative abundance of different classes of chloroplast RNA.